Secondary siRNA screen (5-ethynyl uridine RNA metabolic labelling)

Secondary image-based genetic screen targeting 463 genes in human HeLa cells using RNA interference (siRNA), as described in Berry et al, 2021. Cells incubated with 5-ethynyl uridine (5-EU) for 30min before fixation. Nascent RNA detected by click reaction with AlexaFluor488-azide. Other stains: DAPI (DNA), proliferating cell nuclear antigen (PCNA), succinimidyl ester AlexaFluor647 (total protein). Cells fixed 72h after siRNA transfection. Imaged at 20X magnification with two experimental replicates per condition. Further description available in Berry et al., 2022. Single-cell features provided as background-subtracted mean and sum intensity values for the nucleus, cytoplasm, or whole-cell, as appropriate, for each fluorescence channel. Cell-cycle (G1/S/G2) annotation using a classifier trained using PCNA and DAPI features on EdU-incubated cells, as described in the manuscript. Data cleanup and correction applied as described in the manuscript. Summaries provided on a per-well basis.